The Role of Epigenetic Modifiers, Kdm6a and Kdm6b, in Calvarial Suture Development and Craniosynostosis

Abstract

The five flat bones of human calvaria are held together by fibrous sutures, which remain open during development to accommodate for the growing brain. However, excessive osteogenic differentiation of mesenchymal progenitor cells (MPC) within the sutures can lead to premature suture fusion or craniosynostosis. Saethre-Chotzen Syndrome (SCS) is a common form of craniosynostosis, caused by TWIST-1 gene mutation. Currently, the only treatment for craniosynostosis involves multiple invasive cranial surgeries, which could lead to serious complications. Thus, an attempt to identify a non-invasive therapy is paramount. The present thesis has shown that the expressions of histone demethylases, KDM6A and KDM6B, are upregulated in calvarial cells from SCS patients and from an SCS model of Twist-1 haploinsufficient (Twist-1del/+) mice. KDM6A and KDM6B have been shown previously to promote osteogenesis in MPC by removing their epigenetic target, the repressive mark of tri-methylated lysine 27 on histone 3 (H3K27me3), from the promoters of osteogenic genes. An established pre-clinical SCS mouse model was utilised to investigate the inhibition of Kdm6a and Kdm6b activity using the pharmacological inhibitor GSK-J4 on calvarial cell osteogenic potential. Furthermore, a suture mesenchyme-specific deletion of Kdm6a was established to assess the effects of Kdm6a loss, in the suture development of naturally fusing suture, the posterior interfrontal suture. The results demonstrate for the first time that GSK-J4 treatment inhibited the osteogenic potential of calvarial stromal cells in vitro and the bone formation of ex vivo explants of Twist-1del/+ calvaria, with minimal level of toxicity. ChIP analyses revealed that GSK-J4 treatment elevated the levels of the H3K27me3 mark on osteogenic genes leading to repression of their expression. In vivo studies showed that the local administration of GSKJ4 onto the calvaria of Twist-1del/+ prevented premature suture fusion and kept the sutures open throughout calvarial development (Shpargel et al. 2017). Thus, the inhibition of Kdm6a and Kdm6b activity by GSK-J4 could be a potential therapeutic strategy for preventing craniosynostosis in children with SCS. Furthermore, conditional knockout of Kdm6a prevented suture fusion of posterior interfrontal suture, suggesting that Kdm6a involvement could also contribute to non-syndromic craniosynostosis.